Bone Development
Endochondral ossification is a fundamental mechanism for bone formation, whereby cartilage is replaced by bone. Endochondral ossification requires the sequential formation and degradation of cartilaginous structures that serve as molds for the developing bones. The process of endochondral ossification in the cartilaginous growth plate, which is located at both ends of vertebrae and long bones, determines longitudinal bone growth.
During fetal life and until the end of puberty, longitudinal bone growth takes place via endochondral ossification of the growth plate located at the epiphyses (ends) of long bones. The growth plate is divided into several zones of cartilage forming cells, or chondrocytes, with distinct patterns of gene expression. In the Reserve Zone, cells are small and relatively inactive. In the adjacent Proliferative Zone, chondrocytes proliferate, arrange themselves in columns and eventually undergo hypertrophy. In the lower Hypertrophic Region towards the cartilage-bone junction, cells are big and highly active but exhibit no further cell division. The matrix surrounding the hypertrophic cells calcifies and the lowermost cells undergo programmed cell death. Cell death is accompanied by the removal of the cartilaginous matrix and its replacement by bone through the concerted action of recruited bone cells, namely osteoclasts and osteoblasts.
Signaling Pathways in Bone Development
The process of endochondral ossification is the result of the concerted action of several signaling pathways. The signaling pathway triggered by activation of Fibroblast Growth Factor (FGF) receptors have been shown to be involved in several stages of limb and bone development. A number of birth defects are associated with mutations in the genes encoding FGF receptors (FGFR). For example a mutation in FGFR1 is associated with Pfeiffer syndrome. Certain other mutations in FGFR2 are associated with Crouzon, Pfeiffer, Jackson-Weiss, Apert or Beare-Stevenson syndromes. The clinical manifestation of Apert syndrome (AS) is characterized by both bony and cutaneous fusion of digits of the hands and the feet. Broad thumbs and halluces distinguish Pfeiffer syndrome, while in Crouzon syndrome limbs are normal but a high degree of proptosis is evident. The most prominent malformation syndrome associated with these mutations is craniosynostosis (the premature fusion of the skull bones sutures).
FGFR3 has an inhibitory role in bone elongation as demonstrated by the fact that mice lacking this receptor exhibit a phenotype of skeletal overgrowth. Moreover, mutations at various positions in this receptor result in skeletal dysplasias (SD). Thanatophoric dysplasia is a severe and lethal form, while hypochondroplasia is a milder form than Achondroplasia. Examination of the sequence of FGFR3 in Achondroplasia patients identified a mutation in the transmembrane domain of the receptor (reviewed in Vajo et al. (2000) Endocrine Rev 21:23-39).
Achondroplasia is the most common form of short-limbed dwarfism occurring with a frequency of 1:20,000 live births. Patients show characteristic shortening of proximal long bones (rhizomelia), relative macrocephaly, depressed nasal bridge and lumbar lordosis.
Achondroplasia is mainly caused by a Gly380Arg (G380R) mutation in the transmembrane domain of the FGFR3 and is transmitted in an autosomal dominant fashion (Shiang et al. (1994) Cell 78: 335-342 and Rousseau et al. (1994) Nature 371: 252-254). A Gly375Cys (G375C) mutation has also been reported in some Achondroplasia patients. These mutations affect the process of endochondral ossification by inhibiting proliferation and delaying maturation of chondrocytes in the growth plate cartilage of long bones, resulting in decreased elongation.
Other major regulators of bone growth include growth hormone (GH, reviewed in Kelly et al., (2001) Horm Res;55 Suppl 2:14-7); insulin-like growth factor 1 (IGF-1, reviewed in McCarthy and Centrella (2001) Growth Horm IGF Res 11:213-9), glucocorticoids (GC) thyroid hormone (TH, Harvey et al., (2002) Mol Genet Metab 75:17-30) and Vitamin D (van Leeuwen et al, (2001) Steroids 66:375-80).
Each of these molecules exerts its function by binding to specific cell-surface or nuclear receptors of skeletal cells.
Natriuretic Peptides
Natriuretic peptides are known for their role in cardiovascular homeostasis, diuresis, natriuresis and vasodilation. Four isoforms constitute this family: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and dendroaspis natriuretic peptide (DNP). While ANP and BNP are circulating peptides produced by the atria and the ventricle respectively, CNP is hardly found in circulation and is mainly produced in the brain, in vascular endothelial cells and other tissues where it is supposed to work in an autocrine/paracrine manner (Chen and Burnett (1998) J. Cardiovasc. Pharm. 32 Suppl 3:S22-8). DNP is present in human plasma and atrial myocardium (Chen et al (2000) Curr Cardiol 2:198-205) and its sequence disclosed (Schweltz et al (1992) JBC 267:13928-32). CNP from different species have been disclosed in U.S. Pat. No. 5,336,759 (frog); U.S. Pat. No. 5,338,759 (chicken); U.S. Pat. No. 5,973,134 (rat); U.S. Pat. No. 6,020,168 (pig) and U.S. Pat. No. 6,034,231 (human).
Natriuretic peptides effect their biological role through two receptors: NPR-A and NPR-B. These receptors have cytoplasmic guanylyl cyclase domains, which are activated upon ligand binding and lead to accumulation of intracellular cGMP. Some of the effects of cGMP are mediated through two known protein kinases: cGMP-dependent protein kinase I and II. The peptides bind the receptors with different affinities: ANP≧BNP>>CNP for NPR-A and CNP>ANP≧BNP for NPR-B. The tissue distribution of each receptor is different. While NPR-A is expressed in vasculature, kidney and adrenal glands, NPR-B is mainly expressed in the brain.
NPR-C, a third receptor devoid of the kinase and cytoplasmic GC domains is generally considered to be a clearance receptor for removing natriuretic peptides from the circulation, though some other biological functions have been attributed to it (Murthy and Makhlouf (1999) JBC 274:17587-92). This is a widely distributed receptor expressed in almost all the tissues that express a guanylyl cyclase receptor. U.S. Pat. No. 5,846,932 discloses potent ANP variants having decreased affinity for the human clearance or C-receptor. These ANP variants exhibit natriuretic, diuretic and vasorelaxant activity but have increased metabolic stability, making them suitable for treating congestive heart failure, acute kidney failure and renal hypertension. Furthermore, WO00/61631 discloses novel pentapeptide antagonists of the NPR-C.
Natriuretic peptides have a short half life in vivo. In addition to the clearance receptor, they are further cleared from the circulation by degradation. The peptides are cleaved at specific sites, by the neutral endopeptidase 24.11 (NEP) which is found in endothelial cells covering the vascular walls. Human BNP is more resistant to this degradation while ANP and CNP are readily degraded by this enzyme. Inhibition of NEP by inhibitors, including the compounds thiorphan or candoxatril (Ohbayashi et al. (1997) Clin. Exp. Pharma. Physiol. 25: 986-91; Brandt et al. (1997) Hyperten. 30: 184-90), increases the concentration of endogenous or administered peptides in the circulation.
CNP, like ANP, BNP and DNP, was shown to exhibit natriuretic and hypotensive actions. Novel CNP-related peptides capable of eliciting a strong cGMP response and suppressing the growth of vascular smooth muscle cells have been disclosed in U.S. Pat. No. 5,434,133. Also disclosed are the amino acids responsible for the cGMP producing activities and novel CNP variants capable of inhibiting abnormal growth of smooth muscle cells, for the treatment of, inter alia, restenosis and arteriosclerosis.
Transgenic mice, over-expressing BNP show a skeletal phenotype characterized by overgrowth of the axial and appendicular skeleton (Suda et al. (1998) PNAS 95: 2337-42). Moreover, mice that are null mutants for the clearance receptor, NPR-C, exhibit similar skeletal overgrowth, consistent with a role for the local modulation of natriuretic peptides levels by NPR-C (Matsukawa et al. (1999) PNAS 96: 7403-08). CNP and its specific receptor, NPR-B, have been shown to be expressed in the proliferating zone of the growth plate in fetal mouse tibia while NPR-C has been shown to be expressed in the region of hypertrophic chondrocytes and in osteoblasts (Yamashita et al. (2000) J Biochem 127: 177-9). After the date of the present invention, Chuso et al (Chusho et al. (2001) PNAS 98:4016-21) have disclosed CNP knockout mice which exhibit skeletal phenotypes histologically similar to those seen in A chondroplasia mice. They also reveal the rescue of the CNP knock out skeletal defects by tissue-specific ectopic CNP expression in the growth plate. Moreover, ex vivo experiments (fetal bone organ culture) from wild type animals have shown that CNP, more than BNP and ANP, can induce bone elongation (Yasoda et al. (1998) JBC 273: 11695-700, Mericq et al. (2000) Ped Res 47: 189-93).
While much is known about the components of signaling pathways that contribute to the process of endochondral ossification, little is known about the complex interactions between them that coordinate longitudinal bone growth.